PCF REP 3 Total RNA
RNA from T. brucei PCF and BSF were treated with 50% hydrazine for 45 min on ice, ethanol precipitated, washed twice with 70% ethanol, dried and resuspended in 1 M aniline (pH 4.5, adjusted with glacial acetic acid). The samples were resuspended well in ice by pipetting until the RNA pellet dissolves and incubated at 60C for 15 min in dark. The RNA was later recovered by ethanol precipitation and dissolved in ultra-pure water. The treated RNA was then used for library preparation. Briefly, 800ng of the RNA was dephosphorylated with FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific) and cleaned by Agencourt RNA clean XP beads (Beckman Coulter). The RNA was then ligated to 3 linker using high concentration T4 RNA Ligase 1 (NEB) in a buffer containing DMSO, ATP, PEG 8000 and RNase inhibitor (NEB) for 1.5 hr at 22C. The ligated RNA was purified from excess linker using Dynabeads MyOne SILANE beads (Thermo Scientific), and first strand cDNA was prepared using the AffinityScript Reverse Transcriptase enzyme (Agilent) at 55C for 45 min. Next, the RNA was degraded using 2 l of 1 M NaOH, and the cDNA was cleaned using Dynabeads MyOne SILANE beads (Thermo Scientific). The cDNA was further ligated to 3 adapter using a high concentration T4 RNA Ligase 1 (NEB) overnight at 22C and cleaned of excess adapter by using Dynabeads MyOne SILANE beads (Thermo Scientific). The adapter ligated cDNA was PCR enriched using NEBNext High-Fidelity (NEB) polymerase (9 PCR cycles), separated on an E-Gel EX agarose gel (Invitrogen) and size selected for the range of 150300 bp (containing ~30-180nt corresponding to RNA). The amplicons were gel purified using NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel), and sequenced in a Nextseq system (Illumina) in paired end mode (2040 million reads for each sample).